Protein-nucleic acid interactions play an essential role in the expression and regulation of genetic information. We propose to examine the role of RNA conformation, RNA-protein recognition and protein-protein interactions in RNA replication. The study will center on the vitro replication of Q-beta coliphage RNA and related templates by the virally induced Q-beta replicase. This enzyme contains four subunits: a virally coded polypeptide and three host cell protein-elongation factors EFTu and EFTs and the 30S ribosomal protein S1. Replication of Q-beta RNA requires an additional host protein, called host factor. Since four of the five proteins are capable of binding RNA, the functionally important RNA binding sites as well as the nucleotide triphosphate sites and protein-protein contacts will be determined by cross-linking, affinity labeling and chemical modification. We will characterize the binding reactions of the individual components by stopped-flow and temperature-jump relaxation kinetics, and singlet-singlet energy transfer, monitoring either naturally occurring chromophores or fluorescent probes. The basic catalytic activity of replicase and its minimal templates requirements will also be delineated. We will then determine how the elementary reactions and activities are utilized and modulated in the multicomponent machinery that replicates Q-beta viral RNA. Questions to be answered are: a) what is the molecular basis of the stringent specificity replicase shows for its own viral RNA; b) how does replicase produce biologically viable single-stranded RNA products and prevent formation of a biological inactive but more stable duplex of product and template; (c) what are the roles of the host subunits and host factor in RNA replication? The hope of answering these questions and thus the power and appeal of the Q-beta system lies in the ability to manipulate both the RNA and protein components. Although this is a bacterial system, the information gained will facilitate our understanding of gene expression and replication in mammalian systems. Such detailed knowledge is, in term, clearly vital if we are to understand both normal cell development and aberrant cellular processes.